My research aims at understanding the regulation of eukaryotic gene expression, particularly the role played by chromatin architecture in transcription. I propose to employ the Drosophila heat shock response to examine the differences in protein content and physical structure of transcriptionally active and inactive chromatin a step toward more detailed in vitro analyses of eukaryotic transcription. Drosophila melanogaster will be used for this research because of its many synergistic advantages included well-defined genetics, small genome size and the existence of techniques for localizing RNA and protein sequences in polytene chromosome. In particular, the heat shock response of Drosophila cells offers a powerful model system for my studies since heat treatment induces highly selective yet vigorous transcription at a small number of chromosomal sites in Drosophila melanogaster. Recombinant plasmids carrying heat shock genes and adjacent sequences already exist and will be used as probes in studies described below. Specifically, I plan to isolate Drosophila nonhistone proteins probably associated with active genes, namely, histone-binding proteins, HMG-type proteins and cyclic GMP binding proteins. Antisera will be prepared to these proteins to ascertain their chromosomal location ( in "puffed" or active regions?) and to aid in the enrichment for chromatin fractios containing active genes. Nuclease methods (Micrococcal Nuclease DNase I, DNase II) combined with differential solubility and sucrose velocity gradient centrifugation will be used to obtain the initial enrichment for active chromatin. It is hoped that the different lines of experiments will converge to amplify the degree of enrichment. The limits to altered chromatin structure will be probed with plasmids carrying the heat shock genes. The methods of active chromatin fractionation will be tested as a means of enriching for DNA sequences of interest for cloning.